CN102223895B - Method for production of pH stable enveloped viruses - Google Patents
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Abstract
The present invention provides a method for producing pH-stable enveloped viruses wherein said viruses are used for infection of host cells under low pH conditions and for incubation with cell culture cells under conditions of low pH, as well as influenza viruses obtainable by this method which exhibit a high growth rate in cell culture, increased pH and temperature stability and which have human receptor specificity.
Description
Invention field
The invention provides a kind of method for the production of the stable enveloped virus of pH, wherein said virus under low pH condition together with cell culture cell incubation.This viroid comprises pH and the temperature stability of high growth rates, the rising of showed cell in cultivating and has the specific new type influenza virus of human receptor.
Background of invention
In order to prevent by the popular disease causing of annual viral infection, vaccination is most important public health measure.The effective supply of vaccine depends on the ability of a large amount of vaccine raw materials of quick production (for example virus).The fast Development of vaccine and their availability of enriching are vital resisting in many human and animal's diseases.The delay of production of vaccine and lazy weight can cause problem in the time solving the outburst of disease.
Virus (especially influenza virus) demonstrates in the growth containing in embryo egg the effective production that causes influenza virus particles, and this can be used for attenuated influenza virus vaccine strain production deactivation or that live.Even so, recent years, carried out deep work and set up the virus production system that uses cell culture, because egg based method needs are specific, the stable supply of bioclean egg, this can be a problem in pandemic situation.Technology based on cell is a kind of alternative production technology that does not rely on egg supplier and can start acquisition seed virus.In addition; deactivation type influenza vaccines prepared by the virus of cultivating in comfortable mammalian cell demonstrate the more cross reactivity serum antibody of vaccine of induction ratio egg cultivation and disclose better protection (Alymova et al.; 1998, J Virol 72,4472-7).In addition, according to previous result, after containing growth in embryo egg, the receptor-specific of mankind's isolates and antigenicity characteristic change (Mochalova et al., 2003, Virology 313,473-80; Romanova et al., 2003, Virology307,90-7).
On the other hand, the repeatedly amplification of virus in tissue culture usually causes the pH merging to raise HA mutant (Lin et al., 1997, Virology 233,402-10), this reduces relevant (Ruigrok et al., 1986) with virus to the stability of thermal denaturation.The structure of any protein and its stability are based on noncovalent interaction, as hydrophobic force, Van der Waals interaction, hydrogen bond and ionic interaction.PH threshold (the Rachakonda et al. of the fusion of the protein stability induction that the ionic interaction that the known sudden change occurring in the time that virus adapts to cell culture changes in improving because of HA molecule and salt bridge reduce, 2007, Faseb J 21,995-1002).Conventionally the de-stabilise sudden change seeing in the N end of interface HA1-HA2 or HA2-HA2 district or HA2 can cause (the Korte et al. of the combination to cell surface receptor reducing then, 2007, Rachakonda et al., 2007, Faseb J 21,995-1002, Shental-Bechor et al., 2002, Biochim Biophys Acta 1565.81-9), the immunogenicity that this causes the viral infection of live virus prepared product reduction and reduces subsequently.
Massaab H.F., Journal of Immunology.1969,102, pp.728-732 uses different genetic markers to test at adaptogen for Testis et penis Gallus domesticus tissue culture with before containing the growth in embryo egg and biology and the amynologic characteristic of cold adaptation influenza virus afterwards.Point out that these strains are more responsive to low pH compared with initial " wild type " strain, and shown the remarkable reduction of infectious and hemagglutination output.
Fiszman et al, Journal of Virology, 1974,13, pp.801-808 has checked the impact of low pH (pH6.6) on vesicular stomatitis virus (VSV), and has shown and virion or nucleocapsid do not detected.Ackermann W and Massaab H.F., Journal of Experimental Medicine FEB 1954,99, pp 105-117 has disclosed the impact of viral inhibitors alpha-amido-p-methoxyl group-phenyl methanesulfonamide acid infected by influenza growth cycle.
Owing to being difficult to there is high stability and immunogenicity to avoid the vaccine virus prepared product of any safety or supply problem from a large amount of acquisition of cell culture, an object of the present invention is to obtain and produce effective and stable viral technique.By the application's embodiment is provided, realize this object.
Summary of the invention
The present invention relates to produce the method for the enveloped virus that pH is stable in tissue culture, it has adopted the condition that reduces pH during virus dilution suspension and host cells infected.Method of the present invention also provides a kind of virus, and it is compared with the virion obtaining through the method for current use, and stability and immunogenicity raise.
Accompanying drawing summary
Fig. 1: Wisc. Δ NS1 and Wisc. Δ NS1_HA2_G75R carry out after single intranasal immunity inoculation with 6.0log TCID50/ animal in ferret, relative immunity originality.
Fig. 2:
A) blood serum induced antibody in mouse model;
B) challenge virus is bred in the lung of the mice of immunity and concha nasalis.
Fig. 3:
A. the sequence comparison of the HA molecule of initial virus and mutated viruses.By direct mutagenesis, two nucleotide (aa) are replaced to (tt), cause the aminoacid K of the 58th of HA2 subunit to become I.
B.VN1203 and the erythrocytic fusion activity of VN1203K58I viruses and man.
C. after inoculating with VN1203 and VN1203K58I virus immunity, the IgA antibody titer in mice nose washing liquid.
D. after inoculating with VN1203 and VN1203K58I virus immunity, the HAI antibody titer in mice serum.
E.VN 1203 and the infectivity of VN1203K58I virus to mice.
Fig. 4: Vienna/28 and the sensitivity of Vienna/28_HA2_G75R virus to low pH.
Detailed Description Of The Invention
The present invention relates to a kind of method for the production of enveloped virus, be characterised in that it comprises the steps:
A) have between 5.2 and 5.9, preferably between 5.5 and 5.8, virus dilution in the solution of 5.6 pH most preferably from about;
B) with at least one infectious viral particle host cells infected, wherein: wherein i) described virion is added into described cell; And ii) by described cell and described virion between 5.2 and 5.9, preferably, between 5.4 and 5.8, most preferably from about 5.6 pH incubation is to provide Virus/cells complex;
C) cultivate the described host cell through infection with amplicon virus;
D) gather in the crops described virus; And optional
E) purification and/or characterize described virus.
The virus obtaining by implementation method can be used for the viral production of laboratory scale amount and the large-scale production of vaccine virus.
" large-scale production " means at least 200 liters of minimum volume of culture, and preferably at least 500 liters, the preferably production of approximately 1000 liters.
Containing the vaccine prepared product of envelope virus must be have immunogenic, so that enough vaccination to be provided.Especially, deactivation type Pandemic influenza vaccine (as for bird flu) can have lower immunogenicity, and in the mankind, causes protection antibody and reply and need higher dosage.Effectively antibody response provides the vital immunity for viral infection.Hemagglutinin (HA) albumen is the main target thing of replying by the viral infection of influenza virus and by the protection antibody of the vaccination inductions of deactivation type and work-attenuation type influenza vaccines.It is vital that the structural intergrity of HA antigen is replied for initiation protection antibody.
Inventor has shown that method of the present invention provides the immunogenic virus with pH stability and rising.The HA albumen of the virion so generating preferably shows at low pH the stability raising.Advantageously, virus also can show the stability raising in higher temperature, specifically the high temperature to 60 DEG C.These viruses not have significantly to lose hemagglutination activities of HA, even in the temperature of rising, as 60 DEG C of preservations for example several minutes during to a few hours." not remarkable " means that the reduction of hemagglutination activity compared with the virus of source is less than 4 times.Even after being exposed to the temperature of rising, described virus between between 0 DEG C and 12 DEG C, keep stability after preferably the temperature of 4 DEG C is preserved several weeks to several months.Therefore, for vaccine, preparation is highly favourable to the virus generating according to the present invention, because described virus comprises stable HA molecule.
This method specifically can be used for minus-stranded rna virus, they are a treated animal virus, comprise several important human pathogen, comprise influenza, measles, parotitis, rabies, respiratory syncystial, Ebola (Ebola) and Hantaan virus (hantavirus).
The genome of these RNA viruses can be monomolecular or section, strand (-) polarity.These viruses are shared two key requests: geneome RNA must effectively copy into viral RNA, can be used for mixing the form of progeny virus granule and is transcribed into mRNA, and it translates into virus protein.Eukaryotic host cell does not contain conventionally for replicated rna template or for the mechanism from strand RNA template translation polypeptide.Therefore, minus-stranded rna virus is encoded and is carried RNA RNA-dependent polymerase and carry out the synthetic of the new geneome RNA of catalysis (for being assembled into offspring) and mRNA (for translating into virus protein).
For transmitted virus, genome virus RNA must be packaged into virion.The progeny virus granule assembling occurring between erecting stage is similar with the process of protein/protein interaction in RNA viruses.The formation of virion guaranteed rna gene group from host cell efficient propagation another host cell to same host or different hosts organism.
The Viraceae that contains the genomic peplos single stranded RNA of minus strand is divided into and has genomic group of non-sectionization (Paramyxoviridae (Paramyxoviridae), Rhabdoviridae (Rhabdoviridae), Filoviridae (Filoviridae) and borna's disease virus (Borna Disease Virus), Alphaherpesvirinae (Togaviridae)) or there is genomic group of sectionization (orthomyxoviridae family (Orthomyxoviridae), Bunyaviridae (Bunyaviridae) and Arenaviridae (Arenaviridae)).Orthomyxoviridae family comprises A type, B-mode and influenza virus C, and Suo Getuo (Thogoto) and many reason (Dhori) virus and Infectious salmon anaemia virus.
Preferred embodiment includes but not limited to influenza virus, respiratory syncytial virus (RSV), ewcastle disease virus (NDV), vesicular stomatitis virus (VSV) and parainfluenza virus (PIV).
By containing the genomic internal ribosomal nucleoprotein core of single stranded RNA (helical form nucleocapsid) and inner forming with the outside lipoprotein envelope of stromatin (M1) lining.The section genome of influenza A virus is made up of linearity, negative polarity, the single stranded RNA of 8 molecules, their 11 kinds of (some influenza A strain is 10 kinds) polypeptide of encoding, comprising: RNA RNA-dependent polymerase protein (PB2, PB1 and PA) and form the nucleoprotein (NP) of nucleocapsid; Matrix membrane albumen (M1, M2); Two kinds of surface glycoproteins that protrude from lipid-containing envelope: hemagglutinin (HA) and neuraminidase (NA); Non-structural protein (NS1) and core output albumen (NEP).Most of influenza A strains are also encoded the 11st kind and are thought to have the albumen (PB1-F2) of short apoptosis characteristic.
Genomic transcribe and be replicated in core carry out, and assembling sprouting on plasma membrane carry out.Virus can reprovision gene during mixed infection.Influenza virus is adsorbed to the sialyl oligosaccharide in glycoprotein and glycolipid through HA.After to the endocytosis of virion, in intracellular, there is the conformation change of HA molecule, this promotes film to merge, and so triggers shelling.Nucleocapsid migrates to core, and virus mRNA is transcribed there.Virus mRNA is transcribed by unique mechanism, and wherein viral endonuclease is from 5 '-end of cell allos mRNA cutting belt cap, then serves as virus transcription enzyme and transcribe the primer of viral RNA template.Transcript stops in the site apart from 15 to 22 bases of their template ends, and wherein oligo (U) sequence works to add the signal of poly (A) string.In eight kinds of viral RNA molecules that so generate, six kinds is monocistron information, and they directly translate into the protein that represents HA, NA, NP and varial polymerases albumen PB2, PB1 and PA.Another two kinds of transcripies carry out montage, produce separately two kinds of mRNA, and they translate into different reading frames to generate M1, M2, NS1 and NEP.In other words, 11 kinds of protein of eight kinds of viral RNA sections coding: nine kinds of structures and two kinds of non-structures (NS1 and the recently PB1-F2 of qualification) albumen.
Virus can be selected from natural strain, variant or the mutant of existing; Through the virus of mutation (for example, by being exposed to mutagenic agent, repeatedly going down to posterity. gene and/or go down to posterity in non-permissive host generates); Reassortant (in the virus genomic situation of sectionization); And/or genetically engineered virus (for example use " reverse genetics " technology), they have the phenotype of expectation.
Term " goes down to posterity " and is defined as with specifying virion number inoculation host cell and the described virus of results after given number of days (normally 2-3 days).Virus can have about 2 to 4 and take turns and copy every day.
Known in this field, World Health Organization (WHO) (WHO) is recommended as every year the annual vaccination of carrying out for influenza pandemic and prepares the wild-type virus using in vaccine strain.Then can be by these strains for the production of reprovision vaccine strain, they generally combine the NA of wild-type virus and/or HA gene all the other constant gene segment Cs derivative with donor virus (being often referred to as main donor virus or MDV) from having some desired character.For example, MDV strain can be the temperature sensitive, attenuation of cold adaptation and/or have high growth rates.
According to a specific embodiment, influenza virus is attenuation type influenza virus.Particularly, influenza virus comprises and deletes or modify in the pathogenicity factor of innate immune response that suppresses host cell.For example, attenuation can be derived from cold adaptation Strain or due to the intragenic deletion of NS1 or modification (Δ NS1 virus), and as recorded in WO99/64571 and WO99/64068, by addressing, complete income herein.These viruses are replication defectives, because their copying of interrupting in the respiratory tract of animal.Or virus can comprise deletion or the modification of PB1-F2 gene.
According to the present invention, virus can further comprise the modification of the stability that improves HA molecule in HA gene.For example, Steinhauer et al., 1991, PNAS.88:11525-1152 has identified that the film of the responsible reduction compared with not mutated type virus of K58I sudden change in influenza Rostock's virus (H7N1) HA2 melts pH value.The conformation change of the HA that induced by acid pH of this hint occurs at low 0.7 pH compared with wild-type virus in the mutant form of HA.By this sudden change is introduced to X-31 influenza virus (H3 hypotype), show identical effect.
Term " reassortant (reassortant) " indicator virus in the time mentioning virus comprises from exceeding a kind of parental virus strain or originate derivative heredity and/or polypeptide composition.For example, 7: 1 reassortants comprise that 7 kinds for example, from the derivative viral genome section (or constant gene segment C) of the first parental virus and a kind of complementarity viral genome section from the second parental virus, encode hemagglutinin or neuraminidase.Within 6: 2, reassortant comprises 6 kinds of genome sections from the first parental virus (modal is 6 kinds of internal gene) and 2 kinds of complementarity sections from different parental virus (for example hemagglutinin and neuraminidase).
Particularly, influenza virus vaccine is derived between being very popular or pandemic strains of influenza viruses, for example H1, H3 or B strain.Show that these strains demonstrate the immunogenicity greatly raising in the time producing according to method of the present invention.
According to method of the present invention, can be used for cultivating viral cell can be any desired type, the cell that can cultivate and can be infected by enveloped virus (specifically influenza virus).Particularly, it can be BSC-1 cell, LLC-MK cell, CV-1 cell, Chinese hamster ovary celI, COS cell, muroid cell, human cell, HeLa cell, 293 cells, VERO cell, MDBK cell, mdck cell, CEK (Embryo Gallus domesticus kidney) CEF (chick embryo fibroblast), MDOK cell, CRFK cell, RAF cell, TCMK cell, LLC-PK cell, PK15 cell, Wl-38 cell, MRC-5 cell, T-FLY cell, bhk cell, SP2/0 cell, NS0, PerC6 (human retina cell).
For virus dilution, can use can provide described pH value scope (specifically between pH 5.2 and 5.9, specifically between 5.4 and 5.8) and be physiological any buffer for cell.For example, it can be MES (2-(N-morpholino-ethyl sulfonic acid)) buffer, citrate buffer solution or acetate buffer, specifically uses the buffer based on PBS.In addition, composition can be added into dilute solution, for example salt, as sodium chloride, sodium hydrogen phosphate or potassium dihydrogen phosphate etc.
Term dilution means viral suspension to be diluted to the virion content that is enough to cell to carry out production infection.
According to method of the present invention, infect suitable cell with at least one virion.Those of skill in the art can easily determine the fully needed virion number of infection.With virus infected cell can be specifically with approximately 0.0001 to 10, preferably 0.001 to 0.5 m.o.i. (infection multiplicity) carries out.
Optionally, during dilution step, in dilute solution and/or between infection and/or culture period, can there is macrolide polyene antibiotic or derivant.Particularly, antibiotic is amphotericin B or derivatives thereof.
Particularly, can be before infecting, for example, before infecting about 60-30 minute, adds macrolide polyene antibiotic for 30 minutes before more preferably infecting.
Be used for the optimum antibiotic concentration of viral incubation or cultivation between 0.20 and 0.50 μ g/ml, specifically 0.25 μ g/ml.
Incubation virus makes its temperature in conjunction with cell (especially cell receptor) can be between 20 DEG C and 38 DEG C.The pH of incubation is preferably between 5.4 and 5.8.To dissolve the time in cell in order determining to be enough to allow in virus, can to monitor virus by standard schedule, as by dye marker or electron microscopy.Particularly, in room temperature, the time period is between at least 5 minutes and 60 minutes, preferably between 20 and 60 minutes.
Can add the protease of cutting hemagglutinin precursor protein, and in virus, dissolve in cell can according to the present invention before with influenza infection cell soon, while or carry out soon afterwards.Be to carry out with infection if added simultaneously, protease can be added directly to cell culture to be infected, or for example as the concentrate together with virus inoculation body.Protease is preferably serine protease, particularly preferably trypsin.If use tryptic words, the final concentration adding in culture medium is advantageously 1 to 200 μ g/ml, preferably 5 to 50 μ g/ml, more preferably 5 to 30 μ g/ml.
After infection, further cultivate the cell culture of process infection with replication-competent virus, particularly until the virus antigen of the pathogenic effect of maximum cell or maximum can be detected.Or, can gather in the crops by any time point in the training period.
Can be for example between 6.5 and 7.5 for the pH that cultivates host cell.Depend on the pH stability of the host cell for cultivating for the pH cultivating.This can determine by the viability of testing host cell under condition of different pH.
Term is cultivated and amplification has identical meanings according to the present invention.
For cultivation, any culture medium that can be used for cultured cell is all suitable.Particularly, culture medium can be SFM opti-pro
tMculture medium, a kind of low-protein culture medium of kidney epithelium and relevant cell for culture expression virus.Can between between 20 and 40 DEG C, the temperature cultured cell between 30 and 40 DEG C particularly.
The virus at least generation that can go down to posterity in host cell, but conventionally need number generation, for example three generations at least.
According to a specific embodiments of the method, copy generation influenza virus results and be separated in infect after 2 to 10 days, preferably within 3 to 7 days, carry out.Can be by the method that those skilled in the art will know that from culture fluid separation and harvesting or cell rests thing, for example, by separator or filter.After this, carry out the concentrated of the influenza virus that exists in culture fluid by the method (such as for example gradient centrifugation, filtration, precipitation etc.) those skilled in the art will know that.
Inventor successfully shown, dilutes enveloped virus and infection cell and produce and be presented at the stability of low pH and the immunogenic enveloped virus of rising under low pH condition.These viruses also can be presented at the stability of temperature of rising and/or high growth rates and/or the human receptor's specificity in cell culture.This is surprising, because Scholtissek, 1985.Archives Virol., 85:1-11 has shown that, at low pH value, the infectivity of influenza virus is irreversibly lost.Also point out does not have association between pH and heat stability.
As another embodiment of the present invention, also provide the influenza virus useful as for example seed virus or for the useful virus of vaccination object.Described influenza virus retains detectable hemagglutination activity in the temperature raising, and is retained in 5.4 and 5.8 the stable infectivity of pH scope, has high growth rates and have human receptor's specificity in cell culture.
" seed virus " is defined as the virus of cultivating for inoculating cell.
According to embodiment of the present invention, detectable hemagglutination activity is defined as the hemagglutination activity reduction that is no more than 4 times compared with used source virus.Source virus can be the virus for example directly separating from nose swab.
According to another embodiment, can be provided as the useful virus of vaccine virus.Described virion is stable and demonstrates immunogenicity similar compared with the virus (for example, from Vero cell, MDCK or MDBK cell) obtaining by known cell culture code or that raise at low pH.Particularly, virus shows and the growth rate not being exposed to according to rising compared with the virus of method of the present invention in cell culture.
In addition, virus has temperature stability and has human receptor's specificity.
Temperature stability means hemagglutination activity according to the present invention and grows to time period of 15 minutes in the temperature of height to 60 ° and significantly do not reduce.PH definition of stability be virus 5.6, preferably between between 5.4 and 5.8, the preferred stability of the pH between 5.2 to 5.9.High growth rates means high to 6 log TCID50/ml, preferred growth rate more than 7log TCID 50/ml.
Described virus can obtain by method described in the application.Described virus is particularly useful for vaccine mixture or therapeutic preparaton.In these preparatons, contained influenza virus can be attenuation type virus or deactivation type virus.Deactivation can be implemented by any method known in the art, as killing other agent treated of using in type viral vaccine with formalin or manufacture or processing or be exposed to UV light with non-ionic detergent.The prepared product that comprises influenza virus can be used by any path, as for example subcutaneous, intranasal or intramuscular.
Or the prepared product that contains influenza virus can further comprise known pharmaceutical acceptable carrier or adjuvant strengthens the immunogenicity of used prepared product.
Preferably, through mucous membrane (particularly through intranasal application) is used prepared product, because these viruses have high immunogenicity because of listed feature (being pH stability, temperature stability, high growth rates and human receptor's specificity) above.
Not on the books or indicate before the influenza virus that comprises these features.
Can understand more all sidedly foregoing description with reference to the following examples.But this type of embodiment only represents the method for implementing one or more embodiments of the invention, should not be read as and limit the scope of the invention.
Embodiment
Embodiment 1
Two kinds of influenza strains are built by reverse genetics, it carries from the surface glycoprotein of epidemic strain A type/Wisconsin/67/05 (H3N2) with from all other genes of WHO vaccine strain IVR-116 (reassortant of A type/New Caledonia/20/99 and A type/Puerto Rico/8/34), and lacks the NS gene (Δ NS1) of NS1 open reading-frame.Due to the difference of the Vero cell condition that goes down to posterity, the virus of acquisition differs place's amino acid replacement in the sequence of HA molecule.The first virus, called after Wisc. Δ NS1, it goes down to posterity all the time on Vero cell, and virus inoculation thing first uses the processing of low pH buffer, that is:
Infect buffer (0.1M MES, 150mMNaCl, 0.9mM CaCl at the MES that is supplemented with 0.25 μ g/ml amphotericin B
2, 0.5mM MgCl
2; PH=5.6) in, virus dilution is to suitable moi.With infecting buffer solution for cleaning Vero cell, and virus inoculation thing is applied to cell incubation 30 minutes.Then remove inoculum, and being supplemented with incubation cell in 0.25 μ g/ml amphotericin B and the tryptic serum-free Opti-pro of 5 μ g/ml culture medium.
This method causes the initial viral HA sequence finding in clinical swab to be kept.The second virus increases at neutrallty condition by standard mode, and has obtained a place in HA2 subunit and substitute, i.e. G75R (table 1).Table 1 has shown the viral sequence comparison existing in HA molecule and swab.
Compare the HA nucleotide sequence of the two-strain of cultivating in different condition.Virus Wisc. Δ NS does not obtain any sudden change in HA molecule, and in viral Wisc. Δ NS_HA2_G75R, identifies the place's sudden change G75R that is arranged in HA2 subunit.
Table 1:
Then, compared the stability of two-strain for low pH by method below.In the infection MES of pH scope 5.6-7.5 buffer, virus dilution is specified moi to obtain, and is applied to Vero cell, and incubation 30 minutes is to allow virus infected cell subsequently.After this, remove inoculum, cell is in 37 DEG C of incubation 4-9 hour (depending on strain), then fixing, and detect influenza NP albumen by immunofluorescence.
This test has disclosed viral Wisc. Δ NS1, and to show at pH 5.6 be stable, with the efficiency infection cell identical with neutrallty condition, and mutant virus Wisc. Δ NS1_HA2_G75R completely loses the ability of infection cell at pH 5.6, only at pH 5.8 with the efficiency infection cell (result do not show) identical with neutrallty condition.
After dosage is the single intranasal immunity inoculation of 6.0 log TCID50/ animals, in ferret, compare the immunogenicity of Wisc. Δ NS1 and Wisc. Δ NS1_HA2_G75R virus.The result obtaining proves, according to the measurement of HAI test (GMT 202.9), there is the significantly higher serum antibody titer of viral Wisc. Δ NS1 induction ratio virus Wisc. Δ NS1_HA2_G75R (GMT 27.9) of complete HA sequence, (Fig. 1).
Embodiment 2
Build the strain of two kinds of H1N1 influenzas by reverse genetics, it carries from the surface glycoprotein of epidemic strain A type/Brisbane/59/07 (H1N1) with from all other genes of WHO strain IVR-116, with NS gene (Δ NS1) combination that lacks NS1 open reading-frame.It seems that the virus obtaining differs place's amino acid replacement in the sequence of HA molecule, be because the difference of Vero cell goes down to posterity due to condition.The first virus, called after Brisbane Δ NS1 goes down to posterity in low pH condition under amphotericin B exists.This code causes the maintenance of HA sequence, and this shows as similar with the virus (1st generation) separating in mdck cell from clinical sample.
The second virus, called after Brisbane Δ NS1_HA2_N16I, goes down to posterity by standard method, and has obtained a place in HA2 subunit and substitute, i.e. N16I (table 2).Table 2 has shown the sequence comparison of HA molecule, with initial isolates comparison.
Table 2
Compare the HA nucleotide sequence of the two-strain of cultivating in different condition.Virus Brisbane Δ NS does not obtain any sudden change in HA molecule, and in viral Brisbane Δ NS_HA2_N16I, identifies the place's sudden change N16I that is arranged in HA2 subunit.
The viral Brisbane Δ NS1 that relatively disclosed of the stability of virus to low pH shows as stable.In immunofluorescence assay, be low to moderate 5.6 pH and pH 7.5 and observing the Vero cell of the dyeing of same amount.Mutant virus Brisbane Δ NS1_HA2_N16I less stable, only at pH 5.8 infection cells and do not infect any cell at pH 5.6.With can't see any immunofluorescence signal (result does not show) with the cell of the viral infection of the buffer combination of pH 5.6.
After being the virus of 5.6 log TCID50/ animals to mice single intranasal immunity inoculation dosage, compare the immunogenicity of two-strain.It is higher than Brisbane Δ NS1_HA2_N16I immunogenicity that the result obtaining has disclosed viral Brisbane Δ NS1; induce higher levels of serum antibody (according to the measurement of HAI test); and in the lung of mice and concha nasalis, copying of challenge virus reduced the better protection (Fig. 2 A, B) to animal of instruction.Fig. 2 B specifically discloses the breeding through challenge virus in the lung of immune mice and concha nasalis.
Embodiment 3
Under standard conditions, on Vero cell, cultivate influenza B strain and also cause the appearance of the interface de-stabilise sudden change in HA1-HA2 in HA molecule or HA2-HA2 district, it relates to stability and reduces and then reduce the immunogenicity (data do not show) of mutant virus in animal model.
Embodiment 4
Previously found that the highly pathogenic virus of H5N1 fowl spreading during last decade did not tolerate the mankind's nose washing liquid processing with pH 5.6.They do not tolerate the acidic buffer processing with same pH 5.6 between inoculation Vero cell stage yet.Finding that this instable reason is HA molecular changes conformation to realize the high pH while merging with cell membrane, is pH value 5.6 for H5N1 virus, and for human virus in the scope of 5.2-5.4.
The people such as Steinhauer have proved that the place in H7N7 virus HA2 substitutes, i.e. K58I, can significantly reduce by 0.7 unit by the pH merging.This sudden change is introduced to H3N2 virus and there is similar effect.
By direct mutagenesis, this variation is introduced to A type/VN1203/04 Δ NS1 (H5N1) virus (reassortant, inherit from HA, the NA of A type/VN/1203/04 and M gene and from all the other genes of IVR-116 vaccine, with the Δ NS1 assortment of genes) HA albumen, and be VN1203 HAK58I (Fig. 3 A) by gained viral nomenclature.Fig. 3 A has shown the sequence comparison of the HA molecule of initial and mutated viruses.
Modify the HA of two-strain in trypsin dependency mode.The fusion pH of mutant virus VN1203 HAK58I has reduced by 0.3 unit (data do not show) in the hemolytic experiment that uses human erythrocyte to carry out.
In addition, viral VN1203 HA K58I demonstrates the infectivity loss of reduction at pH 5.6.In mouse immune fluorimetry, after pH 5.6 and 7.5 infects, observe the almost cell of the dyeing of analog quantity with viral VN1203 HA K58I, and in the time that pH 5.6 infects, can't see staining cell (data do not show) by VN1203 virus.
After mice intranasal immunity inoculation, compare the ability of two-strain induce immune response.After immunity inoculation 4 weeks, obtain mice serum and nose washing liquid, and measure HAI and IgA antibody.As shown in Figure 3 B, the IgA antibody titer of VN1203 HA K58I virus induction is higher 4 times than the viral VN1203 with initial HA sequence.
Fig. 3 B has shown after immunity inoculation VN1203 and VN1203 K58I virus, the IgA antibody titer in mice nose washing liquid.
Fig. 3 C has shown after immunity inoculation VN1203 and VN1203 K58I virus, the HAI antibody titer in mice serum.
In order to prove that the HA stability that low pH is raise causes virus better to mammiferous infectivity, build two kinds of similar reassortants, VN1203R and VN1203R HA K58I, it comprises competent (competent) NS gene.The existence of competent NS gene is that in the immune respiratory tract of being competent at organism, effectively viral growth is needed.After each these virus of various dose intranasal vaccination, compare the viral growth of two-strain in upper respiratory tract.Find, compared with the unmodified type VN1203R virus of 4.5log MID50, large 100 times to the infectivity of mice of the viral VN1203R HA K58I with saltant type HA, with MID50 (dosage when mice of the mouse infection dosage-50% is infected) value of 2.5 log grow in upper respiratory tract (Fig. 3 E).
Fig. 3 E has shown after the intranasal infection of various dose, the breeding of virus in mice upper respiratory tract.
Embodiment 5: heat stability
Inoculating amplicon virus with acidity also keeps virus to heat-inactivated stability.By by virus raise temperature incubation after 15 minutes titration viral hemagglutination titre check heat stability.Find to retain hemagglutination activity by the virus (Brisbane Δ NS1 H1N1, Wisconsin Δ NS1 H3N2, Brisbane Δ NS1 H3N2) of low pH inoculated and cultured, even still like this after being exposed to 60 DEG C.The virus (New Caledonia Δ NS1_HA2_113, Wisconsin Δ NS1_HA_218_225_75_81H3N2) of cultivating and obtain the de-stabilise sudden change in HA with standard conditions cannot tolerate the standard processing of 60 DEG C and after 15 minutes, completely lose hemagglutination activity (table 1).
For bird flu virus, discovery contains HA and only has polynary (polybasic) cleavage site to modify the strain of (in trypsin dependency mode) and do not tolerate the even processing of 55 DEG C (Hong Kong 156 Δ NS1 H5N1, VN1203 (6: 2) (H5N1), VN1203 H5N1), in 15 minutes process, completely loses the erythrocytic ability of coagulation.These viral thresholds are 50 DEG C.The sudden change K58I introducing in HA extracellular domain HA2 subunit improves virus stability until 55 DEG C.
Table 3
Human influenza virus Brisbane Δ NS1 H1N1, Wisconsin Δ NS1 H3N2, Brisbane Δ NS1 H3N2, New Caledonia Δ NS1_HA2_113, Wisconsin Δ NS1_HA1_218_225_75_81H3N2 obtain as 6: 2 reassortants, and it carries from the surface antigen HA of corresponding popular virus and NA and from all other genes of IVR-116 strain.IVR-116 (being that WHO recommends the strain for the production of deactivation type vaccine) comprises the surface glycoprotein from A type/New Caledonia/20/99 (H1N1) virus.Virulent NS gene all lacks NS1 open reading-frame.
Avian viruses obtains as 5: 3 (VN1203 H5N1 and VN1203/04 HA K58I H5N1) or 6: 2 (VN1203 (6: 2) H5N1) reassortants, HA, the NA of its succession avian viruses A type/Vietnam/1203/04 or A type/Hong Kong/156/97 and M (in the situation of 5: 3) gene and all other genes from IVR-116 strain.The HA cleavage site of highly pathogenic fowl strain is replaced low pathogenicity avian viruses.All avian viruseses in this research all contain the NS gene that lacks NS1 open reading-frame.
Embodiment 6
6: 2 reassortants of two kinds of influenzas are built by reverse genetics, it contains from the HA of popular viral A type/Vienna/28/06 (H3N2) and NA gene and from all other genes of WHO strain IVR-116, and lacks the NS gene (Δ NS1) of NS1 open reading-frame.It seems that the virus obtaining differs place's amino acid replacement in the sequence of the HA2 of HA molecule subunit, be because the difference on Vero cell goes down to posterity due to condition.The first virus, called after Vienna/28, cultivate, and the second virus (Vienna/28_HA2_G75R) are cultivated with standard conditions under the culture medium of pH 6.5 exists.Virus Vienna/28_HA2_G75R compares in the sequence of HA different from Vienna/28, the HA2 subunit position in initial wild-type virus does not exist G75R.Here substitutes and causes the infectivity of Vienna/28_HA2_G75R virus in low pH reduction compared with the virus of Vienna/28, by precincubation virus in acidic buffer (30 minutes), (Fig. 4) that titration infection titer is measured subsequently.
Embodiment 7
(ovum derives, and derives from NIBSC, UK) goes down to posterity 5 times abreast on MDCK and Vero cell in virus A type/Brisbane/10/2007.Pass after 5 generations, in immunofluorescence assay, the two gained variant is compared to their infectivities at different pH value from initial virus.Obtain data clearly proved initial viral A type/Brisbane/10/2007 pH 5.6 and in neutral pH 7.5 with identical efficiency infection cell.But, to fasten and pass after 5 generations at corresponding cell, two-strain is all lost the ability at pH 5.6 infection cells.Only observe positive cell dyeing at pH 5.8, but in the time using the buffer of pH 5.6, can't see the cell (result does not show) of dyeing.The order-checking of HA gene is disclosed to two kinds of variants and all obtained identical sudden change, be i.e. the 160th D → E in HA molecule.
Embodiment 8
By carrying out continuous passage with two kinds of different modes, i.e. standard (neutrality) conditioned disjunction acidity (infecting at pH 5.6), makes viral A type/East Rennell/3/06 (ovum derives, and derives from NIBSC, UK) adapt to Vero cell.In immunofluorescence assay, the gained virus going down to posterity in neutral pH is increased to 6.7log TCID50/ml by the ability of growing on Vero cell from 4.7log TCID50/ml, but has lost the ability at pH 5.6 infection cells.With with the virus infected cell of the buffer combination of pH 5.6 after do not observe the cell of dyeing, and in neutrality infects, whole monolayer is colored.Viral A type/East Rennell/3/06 that has adapted to the growth on Vero cell reaches titre 7.6 log TCID50/ml in the time using acid condition to infect, and keeps infectious in low pH condition simultaneously.In immunofluorescence assay, pH 5.6 with observe similar staining cell after 7.5 infect and distribute.
Embodiment 9
By the several continuous passage of carrying out at acid condition (at pH 5.6 infection cells), make viral A type/California/7/09 (ovum derives, and derives from CDC) of new H1N1 hypotype adapt to Vero cell.Gained viral nomenclature is A type/California/7/09-acidity.In bioreactor, carry out (10 liters) production on a small scale, subsequent purificn by initial virus with through the virus adapting to.After each production stage, the acid viral yield of A type/California/7/09-of measuring by hemagglutination titre (HA) than the viral high 2-8 in A type/California/7/09 doubly.The results are shown in table 4.
Table 4: the output of the acid virus of A type/California/7/09 and A type/California/7/09-
By both virus harvests, according to the standard schedule purification for purification deactivation type vaccine also relatively.The result obtaining has disclosed viral A type/California/7/09-acidity and in the parameter of all measurements, has been better than A type/California/7/09 (table 5).
Table 5: the comparison of the purification thing of the acid virus of A type/California/7/09 and A type/California/7/09-
Claims (23)
1. for the production of a method for influenza virus, be characterised in that it comprises the steps:
A) virus dilution in the solution with the pH between 5.2 and 5.9;
B) with at least one infectious viral particle host cells infected, wherein:
I) described virion is added into described cell; And
Ii) by described cell and described virion at the pH incubation between 5.2 and 5.9 so that Virus/cells complex to be provided;
C) cultivate the described host cell through infection with amplicon virus;
D) gather in the crops described virus; And
E) optional purification and/or characterize described virus.
2. for the production of a method for influenza virus, be characterised in that it comprises the steps:
A) virus dilution in the solution with the pH between 5.2 and 5.9;
B) with at least one infectious viral particle host cells infected, wherein:
I) described virion is added into described cell; And
Ii) by described cell and described virion at the pH incubation between 5.5 and 5.8 so that Virus/cells complex to be provided;
C) cultivate the described host cell through infection with amplicon virus;
D) gather in the crops described virus; And
E) optional purification and/or characterize described virus.
3. for the production of a method for influenza virus, be characterised in that it comprises the steps:
A) virus dilution in the solution with the pH between 5.2 and 5.9;
B) with at least one infectious viral particle host cells infected, wherein:
I) described virion is added into described cell; And
Ii) by described cell and described virion at approximately 5.6 pH incubation so that Virus/cells complex to be provided;
C) cultivate the described host cell through infection with amplicon virus;
D) gather in the crops described virus; And
E) optional purification and/or characterize described virus.
4. for the production of a method for influenza virus, be characterised in that it comprises the steps:
A) virus dilution in the solution with the pH between 5.5 and 5.8;
B) with at least one infectious viral particle host cells infected, wherein:
I) described virion is added into described cell; And
Ii) by described cell and described virion at the pH incubation between 5.2 and 5.9 so that Virus/cells complex to be provided;
C) cultivate the described host cell through infection with amplicon virus;
D) gather in the crops described virus; And
E) optional purification and/or characterize described virus.
5. for the production of a method for influenza virus, be characterised in that it comprises the steps:
A) virus dilution in the solution with the pH between 5.5 and 5.8;
B) with at least one infectious viral particle host cells infected, wherein:
I) described virion is added into described cell; And
Ii) by described cell and described virion at the pH incubation between 5.5 and 5.8 so that Virus/cells complex to be provided;
C) cultivate the described host cell through infection with amplicon virus;
D) gather in the crops described virus; And
E) optional purification and/or characterize described virus.
6. for the production of a method for influenza virus, be characterised in that it comprises the steps:
A) virus dilution in the solution with the pH between 5.5 and 5.8;
B) with at least one infectious viral particle host cells infected, wherein:
I) described virion is added into described cell; And
Ii) by described cell and described virion at approximately 5.6 pH incubation so that Virus/cells complex to be provided;
C) cultivate the described host cell through infection with amplicon virus;
D) gather in the crops described virus; And
E) optional purification and/or characterize described virus.
7. for the production of a method for influenza virus, be characterised in that it comprises the steps:
A) virus dilution in the solution with approximately 5.6 pH;
B) with at least one infectious viral particle host cells infected, wherein:
I) described virion is added into described cell; And
Ii) by described cell and described virion at the pH incubation between 5.2 and 5.9 so that Virus/cells complex to be provided;
C) cultivate the described host cell through infection with amplicon virus;
D) gather in the crops described virus; And
E) optional purification and/or characterize described virus.
8. for the production of a method for influenza virus, be characterised in that it comprises the steps:
A) virus dilution in the solution with approximately 5.6 pH;
B) with at least one infectious viral particle host cells infected, wherein:
I) described virion is added into described cell; And
Ii) by described cell and described virion at the pH incubation between 5.5 and 5.8 so that Virus/cells complex to be provided;
C) cultivate the described host cell through infection with amplicon virus;
D) gather in the crops described virus; And
E) optional purification and/or characterize described virus.
9. for the production of a method for influenza virus, be characterised in that it comprises the steps:
A) virus dilution in the solution with approximately 5.6 pH;
B) with at least one infectious viral particle host cells infected, wherein:
I) described virion is added into described cell; And
Ii) by described cell and described virion at approximately 5.6 pH incubation so that Virus/cells complex to be provided;
C) cultivate the described host cell through infection with amplicon virus;
D) gather in the crops described virus; And
E) optional purification and/or characterize described virus.
10. according to the method for claim 1 to 9 any one, be characterised in that 60-30 minute before infecting adds macrolide polyene antibiotic or derivant.
11. methods according to claim 10, are characterised in that described macrolide polyene antibiotic or derivant are amphotericin B.
12. methods according to claim 1 to 11 any one, are characterised in that described cell is tissue culture cells.
13. methods according to claim 12, are characterised in that described tissue culture cells is selected from lower group: BSC-1 cell, LLC-MK cell, CV-1 cell, Chinese hamster ovary celI, COS cell, muroid cell, human cell, HeLa cell, 293 cells, VERO cell, MDBK cell, mdck cell, MDOK cell, CRFK cell, RAF cell, TCMK cell, LLC-PK cell, PK15 cell, Wl-38 cell, MRC-5 cell, T-FLY cell, bhk cell, SP2/0 cell, NS0, PerC6.
14. methods according to claim 1 to 13 any one, are characterised in that described virus is A type, B-mode or influenza C.
15. methods according to claim 1 to 14 any one, are characterised in that described influenza virus is attenuated influenza virus.
16. methods according to claim 1 to 15 any one, are characterised in that described influenza virus comprises deletion or modifies in NS1 gene.
17. methods according to claim 1 to 16 any one, are characterised in that described influenza virus is cold adaptation virus.
18. methods according to claim 1 to 17 any one, be characterised in that the pH value of certain limit being provided and being virus dilution in physiological buffer for cell, wherein said buffer is preferably selected from lower group: MES buffer, citrate buffer, and acetate buffer.
19. methods according to claim 1 to 18 any one, are characterised in that and cultivate described cell by culture medium, and preferably SFM opti-pro of described culture medium
tMculture medium.
20. methods according to claim 1 to 19 any one, are characterised in that the described virus at least generation that goes down to posterity in described host cell.
21. by the obtainable influenza virus of method of claim 1 to 20 any one, be characterised in that this virus retains hemagglutination activity in the temperature raising, pH scope 5.5 to 5.8 retains infectious, has high growth rates and preferably have human receptor's specificity in cell culture.
22. comprise the purposes as seed virus according to the enveloped virus granule of the influenza virus of claim 21.
23. comprise the purposes for large-scale production vaccine virus according to the enveloped virus granule of the influenza virus of claim 21 or 22.
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| PCT/EP2009/065812 WO2010060921A1 (en) | 2008-11-25 | 2009-11-25 | METHOD FOR PRODUCTION OF pH STABLE ENVELOPED VIRUSES |
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| CN102858368B (en) | 2010-02-18 | 2017-04-12 | 泰科诺瓦有限公司 | Universal virus-like particle (vlp) influenza vaccines |
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| EP2316923B1 (en) | 1998-06-12 | 2017-10-18 | Icahn School of Medicine at Mount Sinai | Attenuated influenza viruses with altered interferon antagonist activity for use as vaccines and pharmaceuticals |
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| biologic and immunologic characteristic of cold-adapted influenza virus;MAASSAB H F;《journal of immunology》;19690331;第102卷(第3期);摘要，表1 * |
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